Studies on the microsomal mixed-function oxidase system: mechanism of action of hepatic NADPH-cytochrome P-450 reductase.
The mechanism of hepatic NADPH-cytochrome P-450 reductase has been investigated by using a stopped-flow technique. The reduction of the oxidized native enzyme (FAD-FMN) by NADPH proceeds by both one-electron equivalent and two-electron eqiuvalent mechanisms. The air-stable semiquinone form (FAD-FMNH.) of the native enzyme, which is characterized by an absorption shoulder at 635 nm, is also rapidly reduced to another semiquinone form (FADH-FMNH2) by NADPH with the disappearance of the shoulder at 635 nm, but the absorbance change at 585 nm is relatively constant. The FAD moiety in the FMN-depleted enzyme is rapidly reduced by NADPH, and reduced FAD is oxidized in successive one-electron steps by O2 or potassium ferricyanide. These results indicate the possibility of intra-molecular one-electron transfer between FAD and FMN. The rate of cytochrome P-450 reduction decreases in the presence of FMN-depleted enzyme but is nearly restored to the value of the original enzyme with FMN-reconstituted enzyme. These data suggest that FAD is the low-potential flavin, which serves as an electron acceptor from NADPH. On the other hand, FMN, which is the high-potential flavin, appears to participate as an electron carrier in the process of electron transfer from NADPH to cytochrome P-450 during the mixed-function catalytic cycle.
D000818:Animals; D005486:Flavin Mononucleotide / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010743:Phospholipids; D013053:Spectrophotometry; D013552:Swine
D008081:Liposomes; D010743:Phospholipids; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase